Retrovirus vectors are common tools used in gene therapy model systems. However, there are many safety concerns associated with the use of such vectors. A consideration in the construction of retroviral packaging cell lines is the production of high titer vector supernatants free of recombinant replication competent retroviruses (RCRs). In particular, one method to reduce the likelihood of generating RCRs in packaging cells is to divide the packaging functions into two genomes, for example, one which expresses the gag and pol gene products and the other which expresses the env gene product (Bosselman et al., Mol. Cell. Biol. (1987) 7(5):1797–1806; Markowitz et al., J. Virol. (1988) 62(4):1120–1124; Danos & Mulligan, Proc. Natl. Acad. Sci. (1988) 85:6460–6464). This approach minimizes the ability for co-packaging and subsequent transfer of the two genomes, and allows for a significantly decrease in the frequency of recombination to produce RCRs, due to the presence of three retroviral genomes in the packaging cell. High titer retroviral vector stocks have also been generated by a minimal packaging system containing only the gag-pol gene. High titer retroviral HIV-1 vector stocks have also been generated by a minimal packaging system containing only the gag-pol gene and the rev gene. In the event recombinants arise, mutations (Danos & Mulligan, supra) or deletions (Bosselman et al., supra; Markowitz et al., supra) can be configured within the undesired gene products to render any possible recombinants non-functional. In addition, deletion in the 3′ LTR in the vector construct further reduces the ability to form functional recombinant particles.
Several research groups have focused on the lentivirus vector system. Lentiviruses are complex retroviruses that contain, in addition to the common retroviral genes gag, pol and env, other genes with regulatory or structural function. A representative example of a lentivirus is human immunodeficiency virus-1 (HIV-1), an etiologic agent of acquired immune deficiency syndrome (AIDS).
In addition to full-length linear HIV-1 DNA, which serves as the precursor to the integrated provirus, nuclei of virus-transduced cells contain quantities of circular viral DNA forms that do not contribute to provirus integration. These forms have been considered as “dead-end by-products of aborted infections,” with minimum contribution to the virus life cycle.
It is believed that a retroviral vector construct used in the production of recombinant retroviral particles should have two LTRs, each containing a complete R region, because both are believed to be required for efficient vector production and viral replication. The present invention provides a retroviral vector construct containing only one retroviral LTR that is used to produce high titers of recombinant retroviral particles, thereby reducing the likelihood of recombination and rearrangement during particle formation that can lead to the generation of RCR.